1. Field of the Invention
The present invention relates to a method and apparatus for direct coupling of a liquid chromatograph and a mass spectrometer, liquid chromatographyxe2x80x94mass spectrometry, and a liquid chromatographxe2x80x94mass spectrograph.
2. Description of The Prior Art
A mass spectrometer (MS) is a highly sensitive analysis instrument which provides the user with information on the molecular weight and structure of organic compounds and which is therefore indispensable in the fields of organic chemistry, pharmacy and biochemistry. However, MS cannot separate and distinguish the components of a mixture, and therefore when it is a mixture that is to be analyzed, analysis has been difficult. In view of this point, a liquid chromatograph direct coupled mass spectrometer (LC/MS) has been proposed, using a liquid chromatograph (LC) which is superior in separating and distinguishing a mixture, and utilizing the point that non-volatile substances, thermally unstable substances, inorganic and organic compounds, low and high molecular weight substances can be analyzed easily if only they are soluble in solvents.
LC is a device for mixing a sample for analysis with a solvent and separating the mixture under atmospheric pressure, while MS is a device for analyzing an ionized sample in high vacuum. For coupling the two, therefore, it is necessary to remove the solvent (desolvation) from LC effluent, then ionizing the sample remaining after desolvation and feeding the ionized sample to MS held in high vacuum. A technique for coupling LC and MS is described, for example, in Japanese Patent Publication No. 43692/83, in which effluent from LC is nebulized, the resulting mists are desolvated and ionized, and the thus-ionized sample (desolvated effluent) is subjected to mass spectrometric analysis.
Once the effluent from LC is nebulized, the nebulized jet spreads like spray. In this case, mists of relatively large droplets gather at the center of the jet and in the vicinity thereof, while mists of relatively small droplets gather at the marginal portion and in the vicinity thereof. With movement of these mists, the mists of large droplets present centrally and thereabouts are not so greatly influenced by air, etc. because their mass and kinetic energy are large, so that they are not vaporized so much nor do they become so small in droplet diameter. On the other hand, the mists of small droplets present along and near the marginal portion are influenced directly by air, etc. because their mass and kinetic energy are small, so that they become smaller in droplet diameter gradually due to fluid resistance and repeated collision with other droplets, etc. As the droplet diameter becomes smaller, the mists are influenced more greatly by air, etc. and the moving speed becomes lower, that is, the duration of influence becomes longer. As a result, the vaporization of the small droplets present along and near the marginal is promoted more and more to render the droplet diameter smaller.
Thus, when the effluent from LC is nebulized and with movement of the resulting mists, the mists of large droplets present centrally and thereabouts do not vary so greatly in droplet diameter, while the mists of small droplets present along and near the marginal portion become smaller in droplet diameter, so that variations in droplet diameter become larger as a whole.
Also known is the technique of making finer a nebulized effluent (a mixed sample-solvent solution) from a chromatograph and then removing the solvent from the mists. For making droplets fine, there often is adopted a heating method which is simple in structure, merely involving heating droplets using a vaporizer. For example, the vaporizer is constituted by a metallic block containing a heater so as to permit substantially uniform heating, such as a quartz tube with heating wire wound thereon. Nebulized jet (effluent from a chromatography is heated by radiant heat from the vaporizer while it passes through a vaporizing space surrounded with the vaporizer. A marginal flow of the nebulized jet is lower in moving speed than a central flow due to friction with the wall surface of the vaporizer and can be supplied with a larger amount of heat from the wall surface because of its closer position to the wall surface. More particularly, the marginal droplets absorb infrared rays emitted from the surrounding wall surface, whereby the vaporization of liquid from the droplet surfaces is accelerated to promote the micronization of droplets. Most of the infrared rays is consumed for the micronization of mists in the marginal flow and does not reach the central flow of mists. Therefore, the central droplets cannot be heated to a satisfactory extent. Since the mists in the marginal portion are originally small in droplet diameter due to spray-like diffusion and absorb a larger amount of radiant heat, the diameter of the marginal droplets decreases rapidly. Conversely, droplets larger in diameter are concentrated on the central portion. For this reason, the distribution width of droplet diameters in the nebulized jet becomes larger during movement through the vaporizing space than at the time when the mists were generated. Thus, according to the heating method, fine mists gather in the marginal portion, while mists larger in diameter are present centrally in a larger proportion, rather presenting larger variations in the distribution of droplet diameters.
The nebulized effluent is further subjected to desolvation. For example, by the application of heat, only sample is extracted from a mixed sample-solvent solution. If the distribution range (scatter or variations) of droplet diameters is wide, there arises a problem.
Particularly, giant droplets pose a problem. More particularly, if desolvation is performed under severe conditions (overheating and accelerative collision of ions with high energy) for the desolvation of large-diameter droplets, it is indeed possible to extract sample from the mixed solution containing the large droplets, but the droplets smaller in diameter pass the desolvation and undergo thermal decomposition or are converted into fragment ions, which cannot be analyzed. On the other hand, if desolvation is performed under mild conditions (low heat and acclerative collision of ions with low energy) so as to prevent thermal decomposition, etc. of droplets smaller in diameter, the desolvation of the larger droplets becomes insufficient and if they are introduced into MS, they are detected as noise, thus resulting in deterioration of the analytical sensitivity.
It is an object of the present invention to attain a highly sensitive mass spectrometry while avoiding the damage of sample such as thermal decomposition.
In connection with LC/MS interface, atmospheric pressure ionization (API) has come to be used widely. According to API, effluent from a liquid chromatograph is ionized under atmospheric pressure by such as method as electrospray (ESI) for example. The resulting ions are conducted into a chamber called medium pressure chamber. In the medium pressure chamber, upon change in pressure from atmospheric to vacuum, under evacuation using a vacuum pump, the ions introduced into the same chamber undergo adiabatic expansion due to sudden drop of pressure and is cooled rapidly, whereby a polar molecule, e.g. water, is added to the ions to form cluster ions. The cluster ions are desolvated for mass spectrometry. For example, an ion drift voltage is applied to the cluster ions to induce acceleration and repeated collision of the ions with neutral molecules, and the energy of this collision is taken into the interior to remove the added polar molecules (desolvation by collision-induced dissociation).
Unless the cluster ions are desolvated to a thorough extent, a chemical noise will be generated. This chemical noise will be explained below.
Sometimes there appear peaks at equal intervals on mass spectrum. These peaks correspond to chemical noises caused by cluster ions. Particularly, noises induced by cluster ions of water appear in a large number with high intensity. Also, noises sometimes appear due to the addition of water molecules to molecular ions of a sample. In this case, one ion species is dispensed into several ion species, so that the ion current value of the molecular ions which are to be detected becomes lower. The development of such cluster ions can be suppressed by heating mists to a thorough extent in ionization to vaporize sample and solvent to a perfect extent or by heating the whole of the interface. However, if all droplets are to be vaporized by heating, small-diameter droplets will continue to undergo heat for a long time after vaporization. Such excess heating will cause thermal decomposition of the sample molecules, resulting in loss of all information on the molecular weight and structure of the sample, and hence the mass spectrometry, or analysis as LC/MS, of the sample which is to be detected can no longer be effected.
On the other hand, the applied heat is consumed for the vaporization of added molecules from cluster ions and droplets to prevent the rise in temperature of molecules or ions and hence prevent thermal decomposition thereof. Mist is a mixture of droplets and gas, so even if mist is heated, the heat is consumed for the vaporization of solvent from the droplet surface, so that the mist temperature does not rise. Consequently, even a thermally unstable substance fed from LC can be sent stably in the state of mist to an atmospheric pressure ion source. Also for this reason, excess heating of mist must be avoided.
For the purpose of diminishing chemical noises induced by cluster ions and prevent thermal decomposition of sample molecules, a precise temperature control for the heating section has been tried. However, it is necessary to perform a best-point searching operation for each object to be measured and thus the trouble of measurement is enhanced markedly.
Further, various sizes of cluster ions and neutral droplets are included, so in the event giant droplets are included, the desolvation in the intermediate pressure chamber by heating and heat-collision of ions becomes insufficient. If the desolvation is performed under severe conditions (overheating and accelerative collision of ions with high energy), small clusters will be decomposed thermally or transformed into fragment ions though the desolvation of large clusters will be sufficient. And if the desolvation is conducted under mild conditions, large clusters and droplets will not be desolvated to a satisfactory extent. Moreover, if large droplets having electric charge or neutral droplets are introduced into MS, vaporization is performed continually while flying through the intermediate pressure chamber and the mass spectrometric portion and they are detected as the foregoing chemical noises by means of a detector. Besides, a large amount of cluster ions appears in a wide mass area and masks the ions of the sample component to be analyzed. As a result, the noise level rises greatly to the extent that highly sensitive analysis is no longer feasible.
It is another object of the present invention to avoid heating sample molecules to a high temperature and thereby prevent thermal decomposition thereof and provide a mass spectrum of good quality, furthermore, prevent the development of cluster ions and make highly sensitive LC/MS measurement possible.
In connection with LC/MS interface and atmospheric pressure ionization, special attention has been paid to atmospheric pressure chemical ionization (APCI) from the standpoint of wide application range and stability, and APCI has come to be used widely. For example, APCI is described in Analytical Chemistry, Vol. 62, No. 13 (1990), pp. 713A-725A, and Journal of Chromatographic Science, Vol. 29 (1891), pp. 357-366. In APCI, effluent (a mixed sample-solvent solution) from a liquid chromatograph is nebulized under atmospheric pressure and the resulting mists are exposed to corona discharge at a high voltage of about 3 to 5 kV (using a needle electrode for corona discharge), whereby first solvent molecules are ionized. The ions thus produced repeat ion-molecule reaction with sample molecules, and eventually the sample molecules are ionized. The thus ionized sample is conducted to a mass spectrometer in high vacuum and is subjected to mass spectrometric analysis.
When the effluent from the liquid chromatograph is fed to the vicinity of the needle electrode for corona discharge, if there are large variations in droplet diameter of the mists as mentioned above, there is created a complicated flow. Consequently, the mist flow near the said needle electrode varies continually. As a result, the ion-molecule reaction becomes unstable and it becomes impossible to ensure a stable supply of ionized sample to the mass spectrometer.
Also in APCI, heating the mists of effluent from the liquid chromatograph for micronization is effective in accelerating the subsequent ion-molecule reaction. In this case, however, there will occur not only a great difference in density (variations in droplet diameter) but also a difference in temperature of the nebulized jet, as noted previously. If the nebulized jet having such a temperature difference is fed to the vicinity of the needle electrode for corona discharge, the temperature around the needle electrode will vary continually, also resulting in the ion-molecule reaction becoming unstable. Accordingly, the ionized sample can no longer be fed stably to the mass spectrometer and it becomes impossible to make high sensitive analysis.
It is a further object of the present invention to stabilize the mists of effluent to be fed from a liquid chromatograph to a needle electrode for corona discharge, thereby stabilize the ion-molecule reaction of sample and solvent and make highly sensitive LC/MS measurement possible.
According to the present invention, in order to achieve the above-mentioned objects, in nebulizing the effluent from the liquid chromatograph, then desolvating and ionizing the mists and subjecting the ionized sample to mass spectrometric analysis, the mists are stirred.
According to the present invention, in order to achieve the abovementioned objects, in nebulizing and ionizing the effluent from the liquid chromatograph under atmospheric pressure, followed by desolvation and mass spectrometric analysis in high vacuum, the mists are stirred.
According to the present invention, in order to achieve the foregoing objects, in nebulizing the effluent from the liquid chromatograph, then desolvating and ionizing the mists and subjecting the thus-ionized sample to mass spectrometric analysis, a portion of the mists is selected and subjected to desolvation and ionization.
According to the present invention, in order to achieve the foregoing objects, in nebulizing the effluent from the liquid chromatograph, then heating the mists, desolvating and ionizing the heated mists and subjecting the thus-ionized sample to mass spectrometric analysis, there is used a means for conducting the heat from the source of that beating to further heat the mists.
According to the present invention, in order to achieve the foregoing objects, in nebulizing the effluent from the liquid chromatograph under atmospheric pressure, then ionizing the mists by corona discharge, followed by desolvation and mass spectrometric analysis in high vacuum, the mists are stirred.
According to the above constructions, for example by changing the direction of mist flow mechanically, the mists are stirred and the vaporization of central large droplets is promoted for micronization. Particularly, if the temperature of the marginal mist flow is high, the vaporization will be accelerated to a greater extent. At this time, moreover, the marginal and central flows change places with each other and infrared rays radiated from the surrounding heated wall surface are absorbed uniformly and efficiently by the whole of the mists to make the mists finer.
Besides, the stirring of the mists can be done more effectively by a turbulent flow created behind an object placed in the nebulized jet. As a result, the micronization of droplets is promoted not only by the agitation of the mists but also by a mechanical force of the turbulent flow.
Moreover, by once dividing the mists and then stirring them again, it is made possible to make the mists uniform in temperature and finer in size.
By ionizing uniform and fine droplets and thereafter introducing the thus-ionized droplets into MS, the desolvation of cluster ions by accelerative collision-induced dissociation in the medium pressure portion can be done efficiently, whereby chemical noises derived from cluster ions can be diminished to a great extent. As a result, stable and highly sensitive LC/MS analysis can be realized.
Further, the marginal flow of the nebulized jet containing a high proportion of fine droplets and having a high temperature is introduced into an atmospheric pressure ion source for example and is ionized therein. The resulting ions (including cluster ions) are introduced into the medium pressure chamber and desolvation is performed efficiently by accelerative collision-induced dissociation using an ion drift voltage.
That the temperature is high even slightly mitigates the cooling by adiabatic expansion and makes desolvation effective. As a result, chemical noises derived from cluster ions can be diminished greatly and it becomes possible to make highly sensitive analysis. Even if the solvent composition changes every moment in gradient analysis or the like, the desolvation in the medium pressure chamber can be done efficiently because only the micronized marginal portion is ionized and taken in.
Further, for example by finely dividing the nebulized jet mechanically and greatly increasing the area of the heated wall surface, infrared rays radiated from the heated wall surface can be absorbed efficiently up to the interior of the nebulized jet. As a results, the mist droplets can be made finer and uniform and it is possible to attain uniforming of temperature.
Consequently, particularly, the accelerative collision-induced dissociation in the medium pressure portion is done efficiently and chemical noises derived from cluster ions can be diminished to a great extent. As a result, not only stable and high sensitive analysis can be realized, but also stable measurement is ensured also in gradient analysis involving everchanging solvent composition.
Further, for example by changing the mist flow mechanically, the mists are stirred together to accelerate the vaporization of the central droplets for micronization. Consequently, the mist flow fed to the needle electrode for corona discharge becomes stable to stabilize the chemical ionization of sample and solvent, whereby ionized sample can be fed stably to the mass spectrometer. As a result, sensitivity is improved.